The continuation of our studies on the biosynthesis of antibiotics and other biologically active microbial metabolites will combine classical chemical and biochemical with molecular genetic approaches to delineate biosynthetic pathways and mechanisms. Compared to previous work the focus will be on fewer compounds and systems which will be examined in greater detail. The following specific aims will be pursued: 1) To establish in detail the pathway by which the phenazine ring system of the saphenamycins and the diphenazine system of the esmeraldins is derived from the shikimate pathway. 2) To clone, sequence and express the genes coding for the enzymes involved in the formation of the mC7N unit of rifamycin in Nocardia mediterranei, to characterize the gene products and compare them to the normal shikimate pathway enzymes, and to probe for the presence of the pathway genes in other organisms. 3) To isolate key enzymes involved in the formation of cyclohexanecarboxylic acid and the genes encoding them from the asukamycin producer, Streptomyces nodosus subsp. asukaensis, to map out the pathway to the unusual mC7N unit in this antibiotic, and to try to clone and express a putative gene coding for the enzyme catalyzing the intramolecular cyclization of 5-aminolevulinic acid to the C5N unit of asukamycin. 4) To map the gene clusters coding for the biosynthesis of the thiopeptide antibiotics thiostrepton in S. laurentii and nosiheptide in S. actuosus, to assign functions to various genes or regions in the clusters, to isolate and characterize enzymes involved in the pathway from cell-free extracts of the wild-type organism, and in a collaborative study to determine by multidimensional NMR spectroscopy the structures of thiostrepton and nosheptide in solution and bound to a fragment of their receptor, 23S rRNA. These studies will lay the foundation for applications of biosynthetic knowledge in combination with molecular genetic techniques in the metabolic engineering of microorganisms which elaborate new molecular structures of potential medicinal value.